Absolute Protein Quantification by LC/MS^E for Global Analysis of Salicylic Acid-Induced Plant Protein Secretion Responses

Abstract

The plant cell wall is a dynamic cellular compartment consisting of a complex matrix of components that can change dramatically in response to environmental stresses. During pathogen attack, for instance, a wide spectrum of proteins that participate in various sequential processes involved in plant defense is secreted into the cell wall. In this study, a mass spectrometry, data-independent acquisition approach known as LC/MS (E) was used to assess temporal changes in the cell wall proteome in response to different levels of an endogenous inducer of plant disease defense responses, salicylic acid (SA). LC/MS (E) was used as a label-free method that enabled simultaneous protein identification and absolute femtomole quantification of each protein secreted into the extracellular matrix. A total of 74 secreted proteins were identified, 63 of which showed increased specific secretion in response to SA. A majority of this induced secretion occurred within 2 h of treatment, indicating that many proteins are involved in the early stages of plant defenses. We also identified a number of apparently nonclassically secreted proteins, suggesting that, as in many nonplant systems, Golgi/ER-independent mechanisms exist for plant protein secretion. These results provide new insight into plant apoplastic defense mechanisms and demonstrate that LC/MS (E) is a powerful tool for obtaining both relative and absolute proteome-scale quantification that can be applied to complex, time- and dose-dependent experimental designs.

Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)

“…Because all charge states and isotopic peaks of precursor ions are included for fragmentation (15) the LC/MS E technique enables higher sequence coverage and has large advantages in the analysis of highly complex samples consisting of numerous co-eluting peptides (16). Combination of DIA methods with the approach based on Hi3 signal intensities was shown to be of potentially high performance for absolute protein quantification at global scale (17,18) and is therefore used in this study.…”

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

“…Recently, Helm et al (2014) applied the LC-IMS-MS E with Top-3 quantification to quantify the Arabidopsis chloroplast stroma proteome, allowing quantitative modeling of chloroplast metabolism. Two other works used the LC-MS E method to assess the quantitative changes of cytosolic ribosomal proteins in response to Suc feeding and the extracellular proteome in response to salicylic acid (Cheng et al, 2009;Hummel et al, 2012).…”

Proteomic study reveals downregulation of apolipoprotein A1 in plasma of poorly controlled diabetes: A pilot study

“…LC-MS E analysis was performed with 2 µl peptide digest (100 ng/µl concentration) by using nanoACQUITY UPLC online coupled to SYNAPT HDMS system (Waters Corp., Milford, PA, USA) equipped with a nanolockspray ion source with a flow rate of 300 nl/min (external lockmass standard: Glu-fibrinopeptide) as described by Cheng et al (20). Following MS E analysis, data were analyzed with Protein Lynx Global Server software (PLGS, version 2.4, Waters Corp.).…”